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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #293859

Title: Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium

Author
item PARKER, JENNIFER - Auburn University
item WISOTSKY, SARAH - Auburn University
item JOHNSON, EVEN - University Of Florida
item HIJAZ, FARAJ - University Of Florida
item KILLINY, NABILK - University Of Florida
item Hilf, Mark
item DE LA FUENTE, LEONARDO - Auburn University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2013
Publication Date: 1/1/2014
Citation: Parker, J.K., Wisotsky, S.R., Johnson, E.G., Hijaz, F., Killiny, N., Hilf, M.E., De La Fuente, L. 2014. Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium. Phytopathology. 104:15-26.

Interpretive Summary: Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (LAS). Infection with LAS, vectored by the Asian citrus psyllid (Diaphorina citri), is incurable; therefore, knowledge regarding LAS biology and pathogenesis is essential to develop a treatment. However, LAS cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of LAS in vitro, LAS inoculum obtained from seeds of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to two months using quantitative polymerase chain reaction (qPCR) in conjunction with ethidium monoazide (EMA). Media tested were King’s B (K), K with 50% juice from the infected fruit (J50), K with 50% commercially-available grapefruit juice (G50), and 100% commercially-available grapefruit juice (G). Results show that juice-containing media dramatically prolong viability compared to K. Furthermore, qPCR showed biofilm formed at the air-liquid interface of juice cultures contained LAS cells, though next-generation sequencing indicated other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining LAS viability in culture, which will contribute to future development of a culture medium for LAS.

Technical Abstract: Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (LAS). Infection with LAS, vectored by the Asian citrus psyllid (Diaphorina citri), is incurable; therefore, knowledge regarding LAS biology and pathogenesis is essential to develop a treatment. However, LAS cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of LAS in vitro, LAS inoculum obtained from seeds of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to two months using quantitative polymerase chain reaction (qPCR) in conjunction with ethidium monoazide (EMA). Media tested were King’s B (K), K with 50% juice from the infected fruit (J50), K with 50% commercially-available grapefruit juice (G50), and 100% commercially-available grapefruit juice (G). Results show that juice-containing media dramatically prolong viability compared to K. Furthermore, qPCR showed biofilm formed at the air-liquid interface of juice cultures contained LAS cells, though next-generation sequencing indicated other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining LAS viability in culture, which will contribute to future development of a culture medium for LAS.